The present invention relates to snoRNA specific siRNAs capable of downregulating snoRNAs in the nucleolus or the nucleoplasm and, more particularly, to the use of these snoRNA-siRNAs as silencing agents to target nuclear or nucleolar RNA molecules, such as the telomerase RNA.
Small Nucleolar RNAs (snoRNAs)
Numerous families of small RNAs have been discovered, including small nucleoplasmic RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). These small RNA molecules function in mRNA splicing (U1, U2, and U4 to U6 snRNAs), mRNA and rRNA processing (U7 snRNA; U3 and U8 snoRNAs), and site selection for RNA modification by methylation of the 2′ hydroxyl group (box C/D snoRNAs) or by pseudouridine formation (box H/ACA snoRNAs).
The Box H/ACA snoRNAs
The box H/ACA snoRNAs include an ACA trinucleotide sequence located 3-nucleotides upstream of the mature snoRNA 3′ end and a consensus H box sequence (5′-ANANNA-3′), but no other primary sequence identity. Despite this lack of primary sequence conservation, the H and ACA boxes are embedded in an evolutionarily conserved hairpin-hinge-hairpin-tail core secondary structure with the H box in the single-stranded hinge region and the ACA box in the single-stranded tail (Balakin, A., et al., 1996. The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions. Cell 86: 823-834; Ganot, P., et al., 1997. The family of box ACA small nucleolar RNAs is defined by an evolutionarily conserved secondary structure and ubiquitous sequence elements essential for RNA accumulation. Genes Dev. 11: 941-956). Although box H/ACA snoRNAs are associated with higher-order nucleolar structures, little is known about the composition of the presumed box H/ACA snoRNA particles (snoRNPs). Two S. cerevisiae proteins, Gar1p and Cbf5p, have been shown to associate specifically with box H/ACA snoRNAs (Bousquet-Antonelli, C., et al., 1997. A small nucleolar RNP protein is required for pseudouridylation of eukaryotic ribosomal RNAs. EMBO J. 16: 4770-4776). Of these two, only Cbf5p, a putative pseudouridine synthase, is required for H/ACA snoRNA stability (Lafontaine, D. L. et al., 1998. The box H+ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase. Genes Dev. 12: 527-537).
The Box C/D snoRNAs
The box C/D snoRNAs contain two sequence motifs, box C (RUGAUGA, where R is any purine) and box D (CUGA), which are located only a few nucleotides away from the 5′ and 3′ ends, respectively, and generally brought into close proximity by base pairing of the four or five terminal nucleotides. This characteristic 5′-3′ terminal stem-box C/D structure plays a critical role in the control of snoRNA biogenesis and nucleolar localization. C/D box snoRNAs also contain, immediately upstream from box D or from another CUGA motif (box D′) in their 5′ half, 10- to 21-nucleotide regions complementary to rRNA, thereby spanning sites of 2′-O-methylation. In the corresponding snoRNA-rRNA duplex, the 2′-O-ribose methylation is directed to the rRNA nucleotide paired to the fifth snoRNA nucleotide upstream from box D or box D′ (i.e., the +5 rule).
Involvement of snoRNAs in Regulation of Brain Proteins
The range of action of C/D box methylation guide snoRNAs was found to exceed beyond the field of ribosome biogenesis. For example, the brain-specific C/D box snoRNA HBII-52 was found to contain an 18-nucleotide sequence which exhibits a phylogenetically conserved complementarity to a critical segment of serotonin 2C receptor mRNA, pointing to a potential role in the processing of this mRNA. In addition, this snoRNA, together with the HBII-85 snoRNA, although expressed in the brain, were found to be absent from the brains of Prader-Willi (PWS) patients or PWS mouse model, demonstrating a paternal imprinting status (Cavaillé, J. et al., 2000. Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization. Proc. Natl. Acad. Sci. USA. 97: 14311-14316). Moreover, the location of these snoRNAs genes on the PWS-Angelman syndrome (AS) locus at chromosome 15q11-13, may suggest that snoRNA host-genes and even snoRNA themselves may play a role in imprinting. However, since the snoRNAs are located in the nucleolus, there is no easy way to decipher the function of such RNAs in these diseases.
Telomere Replication Involves the Action of Telomerase
The ends of chromosomes have specialized sequences, termed telomeres, comprising tandem repeats of simple DNA sequences. The telomeres protect the chromosomes from fusion, recombination and degradation (McEachern, M J, et al., 2000. Telomeres and their control. Annu. Rev. Genet. 34: 331-358). Due to the discontinuous mode of DNA replication, the normal replication apparatus fails to complete the replication of the DNA at the telomere. Thus, the extension of the telomeric single-stranded 3′ overhangs is catalyzed by a telomerase. Telomerase is a ribonucleoprotein (RNP) enzyme that synthesizes one strand of the telomeric DNA using as a template a sequence contained within the RNA component of the enzyme, designated, the telomerase RNA (TER). The TER subunit of telomerase is comprised of a short sequence which serves as a template for the synthesis of G-rich telomeric repeats and a conserved secondary structure feature that plays essential roles in telomerase assembly and catalytic activity in the nucleolus (Chen, J L, et al., 2000. Secondary structure of vertebrate telomerase RNA. Cell 100: 503-514).
Telomerase RNA Includes an H/ACA snoRNA Domain
The mature human and mouse telomerase RNAs are transcripts of 451 and 397 nucleotides, respectively, including, at the 3′ end, a domain which is remarkably similar in its sequence, structure and function to the snoRNA H/ACA domain (Mitchell, J. R., et al., 1999. A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3′ end. Molecular and Cellular Biology, 19: 567-576). The H/ACA domain confers functional localization of vertebrate telomerase RNAs to the nucleus, the compartment where telomeres are synthesized (Lukowiak A A, et al., 2001. The snoRNA domain of vertebrate telomerase RNA functions to localize the RNA within the nucleus. RNA 7: 1833-44).
Telomere Shortening and Chromosomal Instability
In the absence of telomerase activity, telomeres are shortened by 50-200 nucleotides following each round of chromosome replication (Hastie, N D, et al., 1990. Telomere reduction in human colorectal carcinoma and with ageing. Nature 346: 866-868). Thus, the telomerase replenishes preexisting telomeres and confers their stability. In addition, the telomerase also catalyzes the de novo synthesis of telomeres by the addition of telomeric sequences to chromosome breakpoints that do not end with telomeric repeats.
Telomere shortening has been documented in vivo as a function of human age. However, several human diseases were found to be related to increased telomere shortening and instability. For example, patients suffering from dyskeratosis congenita (DKC) exhibit defects in highly regenerative tissues such as skin and bone marrow, chromosome instability and a predisposition to develop certain types of malignancies. The X-linked form of DKC is caused by mutations in a gene encoding a putative pseudouridine synthase, dyskerin. Dyskerin is associated with H/ACA snoRNAs and with human telomerase RNA. Primary cell lines from DKC-affected males exhibit up to 50% reduction in the human telomerase RNA (hTER) steady state and reduced levels of telomerase activity, suggesting that compromised telomerase function involves in the pathology of the disease (Mitchell, J R, et al., 1999. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 402: 551-555). In addition, the very rare autosomal dominant form of DKC is caused by mutations in the telomerase RNA component (TERC) gene leading to very short telomeres in such patients (Vulliamy, T. et al., 2001. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature 413: 432-435). Moreover, mutations in the TERC gene were also found in patients with idiopathic aplastic anemia and in patients with constitutional aplastic anemia (Vulliamy, T., et al., 2002. Association between aplastic anaemia and mutations in telomerase RNA. Lancet 359: 2168-2170).
Inhibition of Telomerase as Cancer Therapy
Human cell transformation is correlated with the activation of telomerase and telomere stabilization. Thus, it was found that telomerase is activated by the MYC oncogene and that human telomerase reverse transcriptase (hTERT) is involved in cell immortalization via the human Papilloma virus E7 oncogene (Greider, C W. 1999. Telomerase activation. One step on the road to cancer? Trends Genet. 15: 109-112). In addition, the majority of tumors contain active telomerase, whereas most normal cells do not. Moreover, a telomerase knockout mouse has established a link between telomere attrition and chromosomal instability characteristics of human hepatocellular carcinoma (Farazi, P A, et al., 2003. Differential impact of telomere dysfunction on initiation and progression of hepatocellular carcinoma. Cancer Res. 63: 5021-5027).
Thus, inhibition of telomerase activity in tumor cells would lead to telomere shortening and prevention of cancer progression. Therefore, the development of telomerase inhibitors may provide the rational for cancer therapy.
Indeed, several prior art studies have attempted to inhibit telomerase activity. A thio-phosphoramidate oligonucleotide targeted against the telomerase RNA was found to inhibit the cytokine-induced telomerase activity and to induce a progressive telomere shortening [Akiyama, M, et al., 2003. Effects of oligonucleotide N3′EP5′ thio-phosphoramidate (GRN163) targeting telomerase RNA in human multiple myeloma cells. Cancer Res. 63: 6187-6194]. Most recently, a study utilized short siRNAs was able to demonstrate a modest dose-dependent reduction in telomerase activity. In addition, in the same study, transfection of HeLa cells with a plasmid carrying the hTER gene in the sense and anti-sense orientation resulted in silencing of the telomerase RNA (Kosciolek, B A, et al., 2003 Inhibition of telomerase activity in human cancer cells by RNA interference. Mol. Cancer Ther. 2: 209-216). However, these studies resulted in modest inhibition of the telomerase activity. In addition, the use of vectors containing relatively long dsRNA in transfections was most likely involved in inhibition of protein synthesis via the activation of protein kinase R (PKR) and the inactivation of eIF2a.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of downregulating in general and snoRNA in particular devoid of the above limitations.
While reducing the present invention to practice, the present inventors have found that siRNA targeted against snoRNA molecules can silence nuclear and nucleolar RNA molecules containing the box C/D or box H/ACA domains. Thus, the present inventor has generated a method of treating snoRNA-related disorders.